Title: Redox Mechanisms of Ischemia Reperfusion Injury in the Liver Hepatic ischemia/reperfusion injury (I/R) is an important clinical problem associated with orthotopic liver transplantation, hepatic resection, and other surgical procedures that transiently alter hepatic blood flow. During the previous two funding cycles, our research has focused on dissecting the mechanisms by which reactive oxygen species (ROS) modulate signaling pathways following reoxygenation injury in the liver and in cell line models of such injury. This proposal focuses on dissecting two ROS-mediated pathways of NFB activation following liver I/R: reperfusion-initiated c-Src activation and subsequent TNF-initiated IB kinase (IKK) activation. We have found that c-Src-mediated activation of NFB during the acute reperfusion phases of injury is controlled by H2O2-dependent c-Src activation in the endosomal compartment. Our preliminary data on this mechanism suggest that c-Src-mediated NFB activation establishes a TNF-dependent pro-inflammatory program that is detrimental to the liver during later phases of I/R injury. We hypothesize that TNF derived from this c-SrcNFB pathway in hepatocytes acts to amplify pro-inflammatory TNF signaling through Kupffer cells. In addition, we have found that TNF stimulation of NFB also requires the formation of redox-active endosomes to facilitate TNF receptor (TNFR) complex formation and IKK activation. The existence of two independent redox-regulated mechanisms of NFB activation, each associated with the endosomal compartment, prompted us to investigate the sources of ROS required for activation of c-Src and TNFR pathways. Our data suggests that NADPH oxidases (Nox) are the primary source of ROS required for activation of these pathways. This proposal will utilize Nox1-, Nox2-, p22phox-, c-Src-, Rac1- and TNF- deficient mouse models to dissect the redox-dependent components of c-Src- and TNFR-mediated NFB activation following liver I/R and/or hypoxia/reoxygenation (H/R) in isolated hepatocyte and Kupffer cell culture models. The focus of studies outlined in Aim 1 will be to determine the molecular mechanisms that drive the formation of redox-active endosomes during the reoxygenation phase of injury, to facilitate c-Src activation by Nox. Aim 2 will investigate the molecular mechanisms by which endosomal Nox controls TNFR1 activation of NFB, using subcellular fractionation assays and Nox-deficient model systems. In Aim 3 we will dissect how c- Src activation pathways coordinate downstream TNF signaling between hepatocytes and Kupffer cells to establish a pro-inflammatory response in the liver. These studies will provide a fundamental understanding of how the subcellular compartmentalization of ROS facilitates NFB activation in the liver and the manner in which these responses establish a detrimental pro-inflammatory program. Such studies may lead to therapeutic interventions that make it possible to reduce liver injury following I/R by modulating the redox-dependent responses most deleterious to the liver.